Currently, nucleic acid detection technique is a common research means in the field of medicine and biology and has been widely used in qualitative and quantitative measurements. Such nucleic acid detection technique in combination with particularly a nucleic acid amplification method as typified by a PCR method enables detection of the presence of very few cells or microorganisms by targeting a nucleic acid specific to these cells or microorganisms. Therefore, the nucleic acid detection technique has been widely used as a high-sensitivity analysis method for basic research as well as in industry. The nucleic acid detection technique is also utilized for the chasing of distribution channels and authentication by artificially incorporating a nucleic acid of a specific sequence into a material or an article to label it.
On the other hand, a detection method utilizing a nucleic acid amplification method has the problem of interference with analysis of other samples that is caused by amplified nucleic acids produced by nucleic acid amplification reaction during the work of detection. Since the nucleic acid amplification reaction produces an enormous number of molecules of amplified products by the logarithmic nucleic acid amplification, amplified products that are unintentionally present in other samples, reagents, test devices or test environments may be amplified as templates and thereby wrong analysis results may be obtained.
For the purpose of preventing such artificial amplification from amplified products, a means for preventing amplified nucleic acids from interfering with other tests have been developed in addition to general attention to the strict handling of samples, reagents, test devices etc., and the cleaning of test environments. For example, a method of producing an amplified nucleic acid susceptible to uracil-N-glycosylase is known, which method comprises amplifying a nucleic acid in a reaction solution containing deoxyuracil triphosphate. Amplified nucleic acids derived from other tests can be degraded by treating a sample or a reaction solution with uracil-N-glycosylase prior to the amplification reaction. In addition, a method of preventing a nucleic acid from functioning as a template in a nucleic acid amplification reaction is also reported, which method comprises modifying the nucleic acid with a photoactivated psoralen compound via a covalent bond (Non-patent Literature 1).
A technique for modifying a nucleic acid to prevent it from functioning as a template for a nucleic acid amplification reaction has also been used in the detection of microorganisms. It is said that DNA derived from dead cells remains in a sample until several days to three weeks after the cell death. When DNAs are extracted from a sample by a usual procedure, they include DNAs derived from both living cells and dead cells. Thus, for example, when a sample is subjected to a sterilization treatment, the result of sterilization is not fully reflected in a microbial detection method with use of a nucleic acid as an index.
As a solution for the above problem, a method of distinguishing living cells and dead cells which comprises a combination of a nucleic acid-modifying agent with a nucleic acid amplification method has been reported (Non-patent Literature 2, Patent Literature 1). This nucleic acid-based detection method is a method for discriminating living cells from dead cells by using the presence or rate of amplification as an index, which comprises use of a nucleic acid-modifying agent such as ethidium monoazide (EMA) or propidium monoazide (PMA) in combination with a nucleic acid amplification method such as real-time PCR. For example, it is reported that EMA is activated under visible light to be covalently bonded to nucleic acids, so that a nucleic acid amplification reaction is inhibited. At the same time, unbound EMA remaining in a free state in a sample is inactivated by reaction with water molecules. Nucleic acids derived from living cells which EMA has not invaded due to the intact cell walls and cell membranes do not undergo the action of EMA. On the other hand, nucleic acids derived from dead cells which EMA has invaded are modified with the EMA, so that a later nucleic acid amplification reaction is inhibited. Thus, when a sample consisting of a mixture of living cells and dead cells is treated with the above-mentioned nucleic acid-modifying agent, nucleic acids derived from living cells are selectively amplified. To date, such a method has been applied to detection of living cells distinguished from dead cells for many microorganisms such as Escherichia coli O157, Salmonella typhimurium, Listeria, Campylobacter jejuni, and Legionella. However, it has been reported that a high concentration of a nucleic acid-modifying agent gives damage to living cells, while it ensures the binding to nucleic acid derived from dead cells.
Thus, the modification of nucleic acid has a variety of applications. Therefore, a method for modifying nucleic acids more efficiently is demanded.